Oncoviruses and melanomas: A retrospective study and literature review

Abstract The role of human oncoviruses in melanoma has been poorly investigated. The aim of this study was to investigate the association between oncoviruses and melanomas searching for human papillomavirus (HPV), Epstein Barr virus (EBV), and human herpesvirus 8DNA in melanoma specimens. Formalin‐fixed and paraffin‐embedded tissue specimens of cutaneous, mucosal, and ocular melanomas (OM) were selected from the Pathology Departments of the Galliera Hospital (Genoa) and the University Hospitals of Turin and Cagliari. Cutaneous and mucosal nevi have been collected as controls. The oncoviruses search has been performed with different polymerase chain reaction reagent kits. Fifty‐four melanomas (25 mucosal, 12 ocular, and 17 cutaneous) and 26 nevi (15 cutaneous and 11 mucosal) specimens were selected. The detection rate for one of the investigated oncoviruses was 17% in mucosal, 20% in ocular, and 0% in cutaneous melanomas (CMs). Despite the differences between groups seeming remarkable, there was no statistical significance (p > 0.5). Our data do not support a primary role of oncoviruses in melanoma carcinogenesis; however, the finding of HPV and EBV DNA in a considerable fraction of mucosal and OMs suggests that these viruses may act as cofactors in the development of extra‐CMs.


| INTRODUCTION
Melanoma has been classified into subtypes based on the tissue from which the primary tumor arises. Cutaneous melanoma (CM), which arises in non-glabrous skin, is the most common subtype; mucosal melanoma (MM), the rarest subtype, arises from melanocytes of the mucosal membranes; ocular melanoma (OM) develops from the melanocytes in the uveal tract of the eye. 1 The etiology of melanoma is multifactorial, reflecting the interaction between host-related and environmental risk factors. 1 Hostrelated factors consist of peculiar, genetically determined, phenotypic traits such as the light color of the skin, hair, and eye, scarce ability to tan, and propensity to burn, and high number of common and atypical nevi. Genetic susceptibility to melanoma is defined by the presence of germline mutations in high-risk and high penetrance melanoma susceptibility genes, such as cyclin-dependent kinase inhibitor 2A, present in 20%-45% of familial CM cases, and BRCA1-associated protein-1, found in up to 28% of families with CM and OM. 1 With regard to environmental factors, the exposure to ultraviolet (UV) radiation is the main risk factor for CM. 1 Nonsolar occupational risk factors for CM are: exposure to petroleum at automobile manufacturing plants, exposure to benzene and trichloroethylene in the clothing industry and biological/chemical workers and to pesticides in the agriculture industry, farmers and veterinarians. 1 As for MM, since it mostly arises on surfaces which are not exposed to sun, this well-known risk factor for CM is unlikely to be involved. Exposure to formaldehyde for sinonasal MM and cigarette smoking for oral MM have been suggested as risk factors. 2 For OM, besides the host susceptibility factors, two professional occupations, welding, and cooking have been associated with tumor development for the exposure to cooking oil fumes, containing carcinogenic agents such as polycyclic aromatic hydrocarbons, and to intense artificial sources of optical radiation including UV, visible, and infrared light. 3 The role of other environmental factors, such as the human oncoviruses, in the pathogenesis of melanoma has been poorly investigated. Oncoviruses include DNA and RNA viruses that usually are a necessary but not sufficient conditions for developing cancers. 4 Only a few studies have focused on the involvement of human papillomaviruses (HPVs) in melanoma showing contradictory results. [5][6][7][8] The role of other oncoviruses, such as Epstein Barr virus (EBV) and human Herpesvirus 8 (HHV-8), has never been studied.
The aim of this study was to investigate the association between oncoviruses and melanomas of the skin, mucosal membranes, and eye searching for the presence of high risk (HR)-HPVs, EBV, and HHV-8 DNA in melanoma tissue specimens. Archive materials of formalin-fixed and paraffin-embedded (FFPE) melanomas were examined using different polymerase chain reaction (PCR) reagent kits. The same tests have been performed in cutaneous and mucosal nevi, as control samples.

| MATERIALS AND METHODS
A retrospective data search was conducted in the electronic archives of the Pathology Departments of three Hospitals: Galliera Hospital (Genoa) and University Hospitals of Turin and Cagliari.  The specimens with insufficient cellularity for any specific oncovirus analysis were identified as "not suitable" (Table 1).
Tissue sections were retrospectively analyzed by quantitative real-time PCR assays also for DNA search of EBV (RealCycler EBAR-UX/EBAR-GX; Progenie Molecular) and HHV-8 (Real Quality HHV-8; AB Analitica), according to the manufacturer's instructions.

| Statistical analysis
Fischer's exact test was used to analyze the differences in the virus detection rate between melanomas and nevi and between cutaneous and extra-CMs. The results were considered statistically significant at p ≤ 0.05.

| RESULTS
Overall, 54 melanoma specimens (25 MMs, 12 OMs, and 17 CMs) and 26 nevi were selected. Clinical, histopathological, and virological features of the studied specimens were described in Tables 1 and 2.
The HPV detection rate in all melanoma specimens (3/47 suitable cases, 6%) resulted higher than in nevi (0%) but the difference was not statistically significant (p = 0.54); likewise, the HPV detection rate in extra-CMs (3/33 suitable cases, 9%) was higher than in CMs (0%) but the difference was not statistically significant (p = 0.54).
The EBV detection rate in all melanomas (2/39 suitable cases, 5%) resulted higher than in nevi (0%) but the difference was not statistically significant (p = 0.51); similarly, the EBV detection rate in extra-CMs (2/24 suitable cases, 8%) was higher than in CMs (0%) but the difference was not statistically significant (p = 0.51).  Table 3 summarizes the available literature concerning HPV detection in melanoma tissues.
Our study is the first report of melanomas associated with "uncommon" HR-HPV genotypes (pool P4 and P5); indeed, HPV16 and HPV18, the most common genotypes in all HPV-related cancers worldwide, 4 were also those most frequently detected types in melanomas in other studies ( Table 3).
The HPV detection rate in our series (6%) is comparable to that found by Miracco et al. (7%). 16 However, unlike several other studies ( with occasional reactivation and shedding of progeny viruses. Since the lymphoid system is its "natural niche," EBV is a risk factor for the development of B and T cell lymphoproliferative disorders. 20 EBV oncogenesis is primarily driven by latent membrane protein 1 (LMP-1), which acts as a constitutively activated CD40 receptor and is responsible for the transformation of B lymphocytes into proliferating lymphoblastoid cells. LMP 2A and EBV nuclear antigens also contribute to cancerogenesis by modulating key cellular processes. 20 As it has been suggested to explain the presence and the role of EBV in breast cancer, 20 we can assume that EBV-positive lymphocytes may infiltrate the mucosal and ocular tissues and transmit EBV to the melanocytes.
Lastly, in our series, we reported the first case of melanoma that harbored both HR-HPV and EBV DNA. This co-infection has been described in other human cancers (cervical, breast, and prostate cancers), 20  ACKNOWLEDGMENT Open access funding provided by BIBLIOSAN.